Although it has been clear for many years that fibronectin (FN) 1 is alternatively spliced, the functions of, and receptors for, two alternatively spliced segments termed EIIIA (or ED-A) and EIIIB (or ED-B) segments have remained elusive. More is known about a non-homologous IIICS repeat encoding the CS-1 segment, which is a cell adhesive site and ligand for integrin ␣ 4 ␤ 1 (1). Both the EIIIA and EIIIB segments are homologous FN type III repeats and are prominently expressed during embryogenesis; homozygous mutations in FN are embryonic lethal (2-7). During wound healing (5, 8), lung, liver, and kidney fibrosis (9 -11), vascular intimal proliferation (12, 13), vascular hypertension (14), and cardiac transplantation (15), the expression of FNs containing the EIIIA and EIIIB domains is significantly increased. A ϳ170-kDa species of EIIIA-containing FNs is found in synovial fluid from patients with rheumatoid arthritis but not osteoarthritis (16). The EIIIB segment has been postulated to have a role in angiogenesis (17). The EIIIA segment has been observed to regulate cell adhesion and proliferation (18 -21). Liver lipocytes and skin fibroblasts differentiate into myofibroblasts when adhering to FNs that include the EIIIA segment (10, 22). One monoclonal antibody (IST-9) to the EIIIA segment has been shown to inhibit myofibroblast differentiation, whereas another (DH1) blocks chondrogenesis during chick development (10,22,23). Moreover, the expression of MMP-9 is regulated by the EIIIA segment in chondrocytes and myelomonocytic cells potentially through toll-like receptors (24, 25).We recently reported detailed epitope maps for functionblocking monoclonal antibodies that bind to the C-CЈ loop of the EIIIA segment (26). The FN type III (FN-III) repeats, of which the EIIIA segment is one, exhibit high structural homology (27-31) despite only 20 -40% identity in amino acid sequence (32). The canonical FN type III repeat is a conserved ␤-sandwich conformation consisting of two ␤ sheets comprising four strands (G, F, C, CЈ) and three strands (A, B, and E) (27). Epitope mapping of the EIIIA segment reveals that functionblocking mAbs interact with the loop between the C and CЈ ␤-strands and the adjacent Ile 43 and His 44 residues are critical to the epitope (26). Given that these monoclonal antibodies blocked EIIIA function we reasoned that the peptide comprising the C-CЈ loop region (EDGIHEL) could encode a sequence that bound cell surface receptors, possibly integrins.The integrins are a family of heterodimeric transmembrane receptors that mediate cell-extracellular matrix and cell-cell interactions (33). One integrin, ␣ 9 ␤ 1 , binds to a peptide sequence within the B-C loop of tenascin-C (34). This sequence (AEIDGIEL) is similar to the EDGIHEL sequence that we identified in the EIIIA segment (26). The ␣ 9 subunit binds unrelated sequences in other ligands including the vascular cell adhesion molecule-1 (VCAM-1) (35), osteopontin (36), the propolypeptide of von Willebrand factor (pp-vWF) (37), tissue transglutamina...

Alpha-mannosidase-II (alphaM-II) catalyzes the first committed step in the biosynthesis of complex asparagine-linked (N-linked) oligosaccharides (N-glycans). Genetic deficiency of alphaM-II should abolish complex N-glycan production as reportedly does inhibition of alphaM-II by swainsonine. We find that mice lacking a functional alphaM-II gene develop a dyserythropoietic anemia concurrent with loss of erythrocyte complex N-glycans. Unexpectedly, nonerythroid cell types continued to produce complex N-glycans by an alternate pathway comprising a distinct alpha-mannosidase. These studies reveal cell-type-specific variations in N-linked oligosaccharide biosynthesis and an essential role for alphaM-II in the formation of erythroid complex N-glycans. alphaM-II deficiency elicits a phenotype in mice that correlates with human congenital dyserythropoietic anemia type II.

We have cloned and expressed two cDNAs encoding the human lysosomal ␣-mannosidase (EC 3.2.1.24) by RT-PCR of human spleen mRNA. This enzyme is required for the degradation of N-linked carbohydrates during glycoprotein catabolism in eucaryotic cells. The shorter of the two cDNAs (3 kilobases (kb)) was found to encode an open reading frame of 2964 base pairs and, when expressed in Pichia pastoris, was found to encode an enzyme that could cleave high mannose oligosaccharides, oligosaccharides isolated from ␣-mannosidosis fibroblasts, and p-nitrophenyl-␣-D-mannopyranoside substrates. In addition, the Pichia-expressed enzyme was inhibited by swainsonine, and had a pH optimum, K m , and V max characteristic of the enzyme purified previously from human liver. The second, larger RT-PCR product (3.6 kb) was found to contain an insertion and a deletion relative to the 3-kb spleen amplimer and encoded a truncated coding region, indicating that it resulted from alternate transcript splicing. No ␣-mannosidase activity could be detected in Pichia transformants containing this coding region, indicating that it did not encode a functional enzyme. Antiserum raised to the recombinant product of the 3-kb ␣-mannosidase cDNA immunoprecipitated lysosomal ␣-mannosidase activity from human fibroblast extracts. Northern blots identified a 3-kb RNA transcript in all human tissues tested, including ␣-mannosidosis fibroblasts, while minor transcripts of 3.6 kb were also present in several adult tissues. Human chromosome mapping of the mannosidase gene confirmed that the functional gene maps to the MANB locus on chromosome 19. Sequence comparisons were made to previously published human cDNA sequences encoding a putative lysosomal ␣-mannosidase (Nebes, V. L., and Schmidt, M. C. (1994) Biochem. Biophys. Res. Commun. 200, 239 -245) and several differences were found relative to the functional lysosomal ␣-mannosidase encoded by the 3-kb spleen cDNA.The lysosomal catabolism of N-glycans on mammalian glycoproteins occurs through the sequential exoglycosidase digestion of oligosaccharides from the non-reducing terminus down to the carbohydrate-peptide core region (1). Included among the hydrolytic activities responsible for this oligosaccharide degradation is a broad specificity exo-␣-mannosidase activity (EC 3.2.1.24) that catalyzes the hydrolysis of ␣1,2-, ␣1,3-, and ␣1,6-mannoside linkages present in complex, hybrid, and high mannose Asn-linked glycans (2). This enzyme is distinguished from other ␣-mannosidase activities by a combination of a low pH optimum (pH 4.5), a broad natural substrate specificity, activity toward the synthetic substrate p-nitrophenyl-␣-mannoside, and sensitivity to inhibition by swainsonine (3, 4). An enzyme activity with these characteristics has been identified and purified from several species sources including Dictyostelium discoideum (5), and a variety of mammalian tissues (6 -11). Metabolic radiolabeling studies have indicated that the human enzyme is initially synthesized as a polypeptide of ϳ110 kDa that is sub...

Golgi a-mannosidase II (a-Mll) is an enzyme involved in the processing of N-linked glycans. Using a previously isolated murine cDNA clone as a probe, we have isolated cDNA clones encompassing the human a-MII cDNA open reading frame and initiated isolation of human genomic clones. During the isolation of genomic clones, genes related to that encoding a-MIl were isolated. One such gene was found to encode an isozyme, designated acMIIx. A 5-kb cDNA clone encoding a-MIIX was then isolated from a human melanoma cDNA library. However, comparison between a-MIIX and a-MIl cDNAs suggested that the cloned cDNA encodes a truncated polypeptide with 796 amino acid residues, while a-MI consists of 1144 amino acid residues. To reevaluate the sequence of a-MIIx cDNA, polymerase chain reaction (PCR) was-performed with lymphocyte mRNAs. Comparison of the sequence of PCR products with the a-MIIX genomic sequence revealed that alternative splicing of the a-MIIX transcript can result in an additional transcript encoding a 1139-amino acid polypeptide. Northern analysis showed transcription of a-MIIX in various tissues, suggesting that the a-MIIX gene is a housekeeping gene. COS cells transfected with c-MIIX cDNA containing the full-length open reading frame showed an increase of a-mannosidase activity. The ca-MIIX gene was mapped to human chromosome 15q25, whereas the a-MIl gene was mapped to 5q21-22.a-Mannosidase (a-M) activities are involved in both biosynthesis and catabolism of N-linked glycans (1, 2). These enzyme activities are present in cells ranging from yeast to human. There are different forms of a-Ms: lysosomal a-Ms are soluble and involved in degradation of N-glycans, endoplasmic reticulum (ER) and Golgi a-Ms are involved in processing of newly synthesized N-glycans, and cytoplasmic a-Ms may be involved in degradation of dolichol intermediates that are not needed for protein glycosylation or oligosaccharides derived from glycoprotein turnover in the ER (1).. Substrate specificities of these a-Ms differ from each other, and Golgi a-MIl specifically hydrolyzes two peripheral mannosyl residues from Manal1-6(Manal --3)Manal ->6(GlcNAcf31--2Mana1 ->3)[Man/31 ->4GlcNAc,lB --4GlcNAc31 ->]asparagine structure. Several a-Ms have been cloned to date. These include Golgi a-MIT (3, 4), ER/cytosolic a-MI (5), two isozymes of Golgi a-MI (6-8), lysosomal a-M (9), Dictyostelium a-M (10), and yeast a-M (11) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

A universal method to separate and quantify 13 phenolic acids (gallic acid, chlorogenic acid, gentsic acid, vanillic acid, caffeic acid, syringic acid, sinapic acid, p-coumaric acid, ferulic acid, anisic acid, rosmarinic acid, salicylic acid, and cinnamic acid) in some compound herbal medicines was established by liquid chromatographic (HPLC). On an Agela XBP-C18 (5 microm, 4.6 mm x 150 mm) column, a multistep binary gradient elution program and a simplified sample pretreatment approach were used in the experiment. For all of the phenolic acids, detection limits ranged around 0.01 mg/L. Linear ranges of higher than 2 orders of magnitude were obtained with a correlation coefficient of 0.9991 to 1. Repeatability was 0.39-2.24% (relative standard deviation, RSD) for intraday, 1.17-3.96% (RSD) for interday, and 0.14-5.33% (RSD) for drug sample analysis. Recovery, tested by a standard addition method, ranged from 83.3% to 104.9% for various trace phenolic acids.

ReviewApplications of nanomaterials in liquid chromatography: Opportunities for separation with high efficiency and selectivity During recent decades, great efforts have been made to improve the chemical stability, selectivity, and separation efficiency of stationary phases in liquid chromatography. Significant progress has been achieved, especially after the introduction of nanomaterials into separation science. This review covers the applications of nanomaterials playing various roles in liquid chromatography. Future possibilities for developing nanomaterial-based stationary phases are also discussed.

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Alternative splicing of the fibronectin gene transcript gives rise to a group of adhesive glycoproteins showing restricted spatial and temporal expression during embryonic development, tumor growth, and tissue repair. Alternative splicing occurs in three segments termed EIIIB, EIIIA, and V. The EIIIA (or ED-A) segment of fibronectin is expressed prominently but transiently in healing wounds coincident with fibroblast expression of an activation marker, smooth muscle cell ␣-actin. A monoclonal antibody (IST-9) to the EIIIA segment blocks transforming growth factor-␤-mediated smooth muscle cell ␣-actin expression by fibroblasts in culture. A second monoclonal antibody (DH1) blocks chondrocyte condensation in chicken embryos. We find that IST-9 and DH1 react with human, rat, and chicken but not with mouse or frog EIIIA, suggesting that His 44 may be important for antibody binding. A series of deletion mutants of rat EIIIA, constructed as glutathione S-transferase fusion proteins, do not react with either IST-9, DH1, or a third monoclonal antibody (3E2 ) is sufficient to restore fully IST-9 binding and much of the activity of DH1 and 3E2. Our findings demonstrate that the function-blocking antibodies, IST-9 and DH1, bind to the Ile 43 and His 44 residues in a conformationally dependent fashion, implicating the loop region encompassing both residues as critical for mediating EIIIA function.The fibronectins (FNs) 1 comprise a group of extracellular matrix proteins that mediate cell adhesion, migration, proliferation, and differentiation (1). FNs play significant roles in embryonic development and are prominent components of the provisional matrix following tissue injury in adults (2, 3). The fundamental importance of the FNs is substantiated by the observation that homozygous mutations in either the FN gene or in the ␣ 5 integrin, a FN-specific receptor, are lethal (4, 5). The FNs are disulfide-linked, dimeric glycoproteins with structural domains that bind cells, collagen, proteoglycans, and fibrin. Each FN consists of homologous repeats, either type I, II, or III. Individual type III repeats within FN exhibit high sequence similarity between species (greater than 90% identity (6)). Despite variations in protein sequence identity between different type III repeats within FN (20 -40% (6)), these repeats show a high degree of structural homology (7-11). X-ray crystallographic studies demonstrate that each type III repeat consists of two ␤ sheets, made up of four strands (G, F, C, CЈ) and three strands (A, B, E) respectively, folded into a ␤ sandwich (7). This structural arrangement is also conserved in other proteins, including growth hormone (12), tenascin (13), neuroglian (14), tissue factor (15), and chitinases (16).Diversity in the FNs occurs by alternative splicing in two type III repeats termed EIIIA (or ED-A) and EIIIB (or ED-B) and one non-homologous repeat called V (or IIICS) (1). The EIIIA and EIIIB segments are either entirely included or excluded, whereas the V region may be included, excluded, or partial...